The miniprojects that I did in my Msc year are listed below:
MINIPROJECT 1: LOCATING PROTEIN-PROTEIN INTERACTIONS USING MODERN CLONING TECHNIQUES: INVESTIGATION OF GPCR SIGNALLING.
Supervisor: Dr Ann Dixon, Chemistry Dept.
GPCRs form the largest group of signal transduction proteins in vertebrates. They are increasingly becoming the targets of more and more potential drugs. Therefore, elucidation of their structure and function is of fundamental importance. With the discovery that mammalian GPCRs can activate G-protein signalling in yeast, yeast GPCRs have become the focus of many studies. In recent years it has become evident that some GPCRs need to dimerize (or even oligomerize) in order to function properly or to be trafficked effectively to the membrane. An accumulation of evidence of GPCR dimers has convinced many that this is the rule rather than the exception. What interactions drive dimerisation are of particular interest. In this mini-project I am looking at two yeast GPCRs: Ste2, the alpha-pheromone receptor from Saccharomyces Cerevisiae, and Mam2, the alpha-pheromone receptor from Schizosaccharomyces pombe. A number of mutagenesis studies on the former protein have shown that the interaction surface driving oligomerization is an over-expressed amino-acid motif located on the first transmembrane domain (TM1) (Overton et al., 2003). A very similar motif exists on the first transmembrane domain of Mam2. We intend to demonstrate whether the TOXCAT assay (Russ and Engelman, 1999) on TM1 of Ste2 can reproduce the results published by Overton et al. and if successful run the assay using just the first transmembrane domain of Mam2 to determine whether this domain is important for dimerization.
MINIPROJECT 2: COMBINED CAVITY RING-DOWN SPECTROSCOPY (CRDS)-SCANNING ELECTROCHEMICAL MICROSCOPY (SECM) AS NEW PROBE OF DYNAMIC PROCESSES IN SUPPORTED LIPID BILAYER MEMBRANES
Supervisors: Dr Stuart Mackenzie and Prof Pat Unwin, Chemistry Dept.
Evanescent Wave Cavity Ring Down Spectroscopy is an extremely sensitive absorbance technique which can be used to probe processes in the condensed phase. The technique involves coupling a pulsed laser source into an optical cavity in which the beam will resonate. The optical cavity consists of two mirrors and a prism in a triangular arrangement. Light entering the cavity is reflected off the mirrors and is totally internally reflected in the prism and so is able to go round and round inside the cavity. After each round trip around the cavity the laser beam will suffer losses in intensity due to diffraction, scattering and absorption due to the medium inside the cavity. There is a further loss to intensity due to absortion from any species which resides in the evanescent field which is produced upon the total internal reflection of the light in the prism. To measure this loss in intensity the amount of light leaking out of the cavity (e.g. out the back of one of the mirrors or reflected off one of the surfaces of the prism) can be measured over time. It turns out that the curve produced is proportional to the total losses of light within the cavity and so by measuring the background losses the absorption at the prism face can be determined. This technique has been used previously to characterise adsorption. It has also been coupled with electrochemistry to understand the processes happening at the electrode. In my project the adsorption of [Ru(bpy)3]2+ to the silica surface of the prism was studied. Although it wasn't quite the project that was initially planned it was interesting all the same and useful to understand the processes going on within the cell.
MINIPROJECT 3: STOCHASTIC EFFECTS IN REACTION NETWORKS
Supervisors: Dr Luca Sbano and Dr Markus Kirkilionis, Maths Institute
Chemical reaction networks have been used to model many processes happening in the cell including genetic and metabolic processes. In the case where all species are assumed to be present in such large numbers that concentrations can be considered normally deterministic models are used. However if this is not the case (for example in the case of the lac operon where only approximately 10 of the lac repressor lacI are present in the cell) clearly a stochastic approach has to be taken. Similarly, if any of the species involved can be in a number of different discrete states (for example a protein may adopt a number of different conformations) this must be treated stochastically. In this project the aim is to study chemical reaction networks in which some of the species involved can adopt multiple discrete states. The intention is to derive the deterministic dynamics that arise starting from a stochastic model at the microscopic level and then take the limit as the numbers of molecules of each species tends to a large number. This kind of approach is thought to give a better understanding of how the microscopic processes determine those at the macroscopic level.
As a part of my mini-project I went to Germany for a week to a series of seminars presented by a number of us from Warwick and by some students at Heidelberg university. My talk was entitled "Dimensional Reduction of Chemical Reaction Networks", notes on which can be found here .
Everyone who attended the Compact Seminar in the Black Forest (big photo)
Overton, M.C., Chinault, S.L., Blumer, K.J., 2003, The Journal of Biological Chemistry, 278(49), 49369-49377
Russ W.P. and Engelman, D.M., 1999, Proc. Natl. Acad. Sci. USA, 96, 863-868
A cartoon of a GPCR sitting in a membrane.
A typical experimental setup for coupling EW-CRDS with SECM.